Bordetella pertussis Commits Human Dendritic Cells toPromote a Th1/Th17 Response through the Activity ofAdenylate Cyclase Toxin and MAPK-PathwaysGiorgio Fedele1., Fabiana Spensieri1., Raffaella Palazzo1, Maria Nasso1, Gordon Yiu Chong Cheung2¤,

John Graham Coote2, Clara Maria Ausiello1*

1 Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanita, Rome, Italy, 2 Division of Infection and Immunity, Institute of Biomedical

and Life Sciences, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, United Kingdom

Abstract

The complex pathology of B. pertussis infection is due to multiple virulence factors having disparate effects on different cell types.We focused our investigation on the ability of B. pertussis to modulate host immunity, in particular on the role played byadenylate cyclase toxin (CyaA), an important virulence factor of B. pertussis. As a tool, we used human monocyte derived dendriticcells (MDDC), an ex vivo model useful for the evaluation of the regulatory potential of DC on T cell immune responses. The workcompared MDDC functions after encounter with wild-type B. pertussis (BpWT) or a mutant lacking CyaA (BpCyaA2), or theBpCyaA2 strain supplemented with either the fully functional CyaA or a derivative, CyaA*, lacking adenylate cyclase activity. As afirst step, MDDC maturation, cytokine production, and modulation of T helper cell polarization were evaluated. As a second step,engagement of Toll-like receptors (TLR) 2 and TLR4 by B. pertussis and the signaling events connected to this were analyzed.These approaches allowed us to demonstrate that CyaA expressed by B. pertussis strongly interferes with DC functions, byreducing the expression of phenotypic markers and immunomodulatory cytokines, and blocking IL-12p70 production. B.pertussis-treated MDDC promoted a mixed Th1/Th17 polarization, and the activity of CyaA altered the Th1/Th17 balance,enhancing Th17 and limiting Th1 expansion. We also demonstrated that Th1 effectors are induced by B. pertussis-MDDC in theabsence of IL-12p70 through an ERK1/2 dependent mechanism, and that p38 MAPK is essential for MDDC-driven Th17 expansion.The data suggest that CyaA mediates an escape strategy for the bacterium, since it reduces Th1 immunity and increases Th17responses thought to be responsible, when the response is exacerbated, for enhanced lung inflammation and injury.

Citation: Fedele G, Spensieri F, Palazzo R, Nasso M, Cheung GYC, et al. (2010) Bordetella pertussis Commits Human Dendritic Cells to Promote a Th1/Th17Response through the Activity of Adenylate Cyclase Toxin and MAPK-Pathways. PLoS ONE 5(1): e8734. doi:10.1371/journal.pone.0008734

Editor: Delia Goletti, National Institute for Infectious Diseases (INMI) L. Spallanzani, Italy

Received October 5, 2009; Accepted December 7, 2009; Published January 15, 2010

Copyright: � 2010 Fedele et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by grants from Istituto Superiore di Sanita (ISS) Italy-USA scientific cooperation agreement (#28C6); Italian Ministry of Health -Ricerca Finalizzata (8ABF/1, 8AGF); and from the Commission of the European Communities, contract #201502 ChildINNOVAC. The funders had no role in studydesign, data collection and analysis, decision to publish, or preparation of the manuscript. No author has any financial, personal, or professional interests thatcould be construed to have influenced the paper.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: clara.ausiello@iss.it

. These authors contributed equally to this work.

¤ Current address: Laboratory of Human Bacterial Pathogenesis (LHBP), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health(NIH), Bethesda, Maryland, United States of America

Introduction

Bordetella pertussis is a Gram-negative coccobacillus that causes

whooping cough, a respiratory disease representing a severe, life-

threatening illness in infants and children and a significant cause of

morbidity in adolescents and adults [1–3]. The incidence of

pertussis is increasing, even in countries with high vaccine

coverage where immunity to pertussis is waning and a growing

number of vaccinated adults have lost protection. Consequently,

Bordetella infection is once again on the rise as a public health

threat [2,4]. Many aspects of pertussis pathogenesis and the

mechanisms involved in protection from the disease are not fully

understood, a situation complicated by the large number of

virulence factors expressed by B. pertussis that exert disparate

effects on different cell types [5,6].

Adenylate cyclase toxin (CyaA) is secreted by B. pertussis and

binds to the cell surface receptor CD11b/CD18 aMb2 integrin

which is expressed on innate immune cells, including macrophages

and dendritic cells (DC) [7]. Within cells, CyaA is able to disrupt

host cellular functions by increasing the intracellular cyclic AMP

(cAMP) concentration [8–10]. CyaA is able to suppress host

antibactericidal activity by inhibiting chemotaxis, phagocytosis,

superoxide production and expression of pro-inflammatory

cytokines, such as TNF-a and IL-12p70, in monocyte/macro-

phages, neutrophils and DC [11–16], thereby promoting bacterial

colonization and persistence [17,18]. Moreover CyaA is also able

to induce apoptosis in macrophages [19,20].

DC are major players in innate and adaptive immunity, as they

express repertoires of pathogen-recognition receptors that recog-

nize specific molecular signatures of pathogens and trigger

antimicrobial defense responses. Activation of DC leads to the

production of cytokines and other immune mediators promoting

the development of specific T helper (Th) responses that play a

critical role in host protective immunity against infectious agents

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[21,22]. We have previously shown that B. pertussis triggers

maturation of human monocyte-derived (MD)DC that efficiently

perform antigen presentation and, although unable to produce IL-

12p70, drive the development of Th1 immunity [23]. In a

subsequent study we demonstrated that IL-12p70 is inhibited

through a block of IL-12p35 expression [15].

In this study we focused on the mechanisms that B. pertussis

employs to escape host immune responses and the role played by

CyaA. The experimental approach involved the analysis of T cell

responses induced by human MDDC cultured with wild type B.

pertussis (BpWT) or an isogenic mutant strain lacking CyaA,

BpCyaA2 [18], and by using fully functional recombinant CyaA

or a derivative lacking adenylate cyclase activity, CyaA*, to

complement the BpCyaA2 strain [24,25]. These approaches

allowed us to demonstrate the induction by B. pertussis of MDDC

promoting a mixed Th1/Th17 polarization and to clarify the role

played by the p38 and ERK1/2 mitogen-activated protein kinases

(MAPK) in this process. The data shed light on the role played by

CyaA in subverting host immune responses by enhancing Th17

immunity and limiting the induction of Th1 effectors.

Results

B. pertussis CyaA Inhibits MDDC Maturation and CytokineProduction

After the encounter with a pathogen, the differentiation

program of MDDC starts with the modification of surface markers

expression [21,22]. In preliminary experiments MDDC were

treated with BpWT or with BpCyaA2 and the ability of MDDC

to internalize the two types of bacteria and the intracellular

survival of internalized bacteria were measured, as reported in a

previous study for BpWT [23]. The BpCyaA2 strain behaved

similarly to BpWT, both strains had a comparable low capability

to be internalized by- and to survive in- MDDC (data not shown).

Phenotypic maturation of MDDC treated with BpWT or with

BpCyaA2 was then assessed. In order to restore adenylate cyclase

activity to BpCyaA2, recombinant CyaA was simultaneously

administered and a genetically inactivated toxin, lacking adenylate

cyclase enzymatic activity, CyaA*, was used as a control. In

previous studies we defined the optimal bacteria:MDDC ratio to

induce MDDC maturation and functions [23] and here, in

preliminary experiments, we finely titrated CyaA and CyaA* doses

in MDDC cultures. We found that a 50 ng/ml dose of CyaA was

optimal to maximize the induction of intracellular cAMP and to

minimize the induction of apoptosis (data not shown). Table 1

summarizes MDDC surface expression of the co-stimulatory

molecule CD80 and the maturation markers CD83 and CD38

induced by the bacteria. Both B. pertussis strains induced significant

up-regulation of phenotypic markers with respect to untreated

MDDC. The BpCyaA2strain was able to induce significantly

higher levels of CD80 and CD83 expression compared to BpWT

strain (p,0.05), confirming and extending previous data [15,23].

The expression of CD38, a newly described maturation marker

involved in the regulation of IL-12p70 [26,27], was also higher in

BpCyaA2 -treated MDDC compared to BpWT -treated MDDC

(p,0.05). Expression of maturation markers declined to levels

similar to those induced by BpWT when CyaA was added to

MDDC cultured with BpCyaA2, but not when CyaA* was used

(Table 1). No relevant modification of expression was found when

the toxins were used alone to stimulate MDDC, indicating that

any LPS contamination of the recombinant CyaA proteins was

very low, confirming the results of the LAL test [24,25], and not

detectable even using MDDC maturation as a functional

biological assay.

These phenotypic data revealed that CyaA was responsible for

the inhibition of CD80, CD83 and CD38 expression in B. pertussis

-treated MDDC, thereby reducing the overall degree of DC

maturation.

DC maturation leads to the production of several regulatory

cytokines pivotal in driving T helper (Th) cell polarization. Here,

we analyzed the role of CyaA in modulation of cytokine

production by MDDC in the presence of B. pertussis. BpWT strain

was unable to induce IL-12p70 secretion, while BpCyaA2

promoted the release of statistically significant levels of IL-12p70

(P = 0.01) (Figure 1), confirming previous data [15,23]. In order to

confirm that CyaA was responsible for IL-12p70 inhibition,

MDDC were cultured with BpCyaA2 in the presence of CyaA or

CyaA*. Addition of CyaA strongly and significantly inhibited IL-

12p70 production with respect to BpCyaA2- and to BpCyaA2+CyaA* -treated MDDC. CyaA* addition did not significantly

affect IL-12p70 induced by BpCyaA2 -treated MDDC (Figure 1).

The combination of IL-23, IL-6 and IL-1b has been shown to

be crucial in driving the differentiation and expansion of Th17

cells, a recently described Th subset involved in infections and

autoimmune disorders [28–30]. In this context, we found that B.

pertussis induced IL-23 as well as IL-1b and IL-6 in MDDC

Table 1. MDDC phenotypical modulation by CyaA toxin after treatment with B. pertussis strainsa.

Stimuli CD80b (MFI) CD83b (% of positive cells) CD38b (MFI)

BpWT 4464 (2.360.2)c 3966 (2.360.4)c 2362 (2.160.2)c

BpCyaA2 76612 (2.260.3)c,d,e 4665 (2.660.8) c,d,e 4265 (2.660.4)c,d,e

BpCyaA2+CyaA 41610 (2.660.2)c 1964 (2.460.1)c 1964 (2.560.4)c

BpCyaA2+CyaA* 87620 (2.460.3)c 4069 (2.560.9)c 4069 (2.660.8)c

none 2062 (2.160.3) 461 (2.160.6) 1162 (2.460.3)

CyaA 2163 (2.560.1) 663 (2.060.2) 961 (2.260.1)

CyaA* 2264 (2.260.2) 261 (2.760.7) 961 (2.160.3)

aMDDC were treated with indicated stimuli for 48 h and analyzed for indicated surface markers associated with the mature phenotype. Isotype-matched antibodieswere used as negative controls and the values are shown in brackets.

bMean value 6 SE of 23 experiments is indicated.cp,0.05 vs none;dp,0.05 vs BpWT;ep,0.05 vs BpCyaA2+CyaA.doi:10.1371/journal.pone.0008734.t001

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(Figure 1). IL-23 levels induced by BpWT were markedly lower

compared to those induced by BpCyaA2 (P = 0.002). The

addition of CyaA to BpCyaA2 -treated MDDC significantly

inhibited IL-23 production, while the addition of CyaA* was

ineffective (Figure 1). Moreover, CyaA, either expressed by BpWT

or exogenously added to BpCyaA2, was associated with reduced

IL-1b and IL-6 production, although without statistically signif-

icant differences. Both B. pertussis strains were able to induce high

levels of IL-10 and the addition of CyaA inhibited IL-10 induced

by BpCyaA2 (Figure 1).

When used alone, CyaA and CyaA* did not induce any of the

cytokines tested with the exception of IL-23, produced at very low

levels not significantly different than those from untreated MDDC,

confirming again that any LPS contamination of the proteins was

undetectable (data not shown).

CyaA Modulates Th1/Th17 Polarization Induced by B.pertussis Treated MDDC

Considering the cytokine profile induced by B. pertussis in

MDDC, we analyzed their capacity to polarize purified T

lymphocytes, and, in particular, the possibility of Th17 induc-

tion/expansion. Figure 2 shows that BpWT -treated MDDC drove

IFNc- and IL-17 -producing Th effector cells. Remarkably, in

view of the findings in the previous section, when T cells were co-

cultured with BpCyaA2 -treated MDDC, a significant decrease of

IL-17 -producing cells was observed, along with a significant

increase of IFNc -producing cells. The addition of CyaA to

BpCyaA2 -treated MDDC reverted the polarization profile to

that induced by BpWT and IL-17 -producing Th cells were

enhanced, while those producing IFNc significantly decreased.

CyaA* did not cause any significant alteration of the polarization

profile induced by BpCyaA2 -treated MDDC (Figure 2). Th2

polarization, assessed by measuring IL-5 release, was significantly

reduced when MDDC were treated with both B. pertussis strains,

with respect to untreated MDDC.

B. pertussis Triggers MyD88 - and Phosphatidyl Inositol 3Kinase -Dependent Pathways in MDDC

The activation of TLR4 and TLR2 by B. pertussis was

investigated by using TLR2- and TLR4- expressing HEK-293

cells [31]. Figure 3 shows that BpWT and BpCyaA2 were able to

induce a potent activation of TLR2, at levels comparable to

Figure 1. Cytokine production. MDDC were either untreated (none)or treated with BpWT or BpCyaA2, the latter in the presence or absenceof CyaA and CyaA* (50 ng/ml ) for 48 h. IL-12p70, IL-23, IL-1b, IL-6 andIL-10 were assessed by ELISA. Values are expressed as mean 6 SE fromnineteen (IL-12p70), eleven (IL-10), six (IL-23, IL-1b and IL-6) indepen-dent experiments performed with MDDC obtained from differentdonors, and expressed as pg/ml of cytokine released. * p,0.05 vs none;up,0.05 vs BpWT; { p,0.05 vs BpCyaA2; 1 p,0.05 vs BpCyaA2+CyaA*.doi:10.1371/journal.pone.0008734.g001

Figure 2. Th polarization. MDDC either untreated (none) or treatedwith BpWT or BpCyaA2 for 48 h were co-cultured with purified T cellsas described in Methods section. On day 12, supernatants werecollected and secreted cytokines were measured by ELISA. Results areexpressed as mean 6 SE of eight independent experiments performedwith MDDC and T cells obtained from different donors. * p,0.05 vsnone; u p,0.05 vs BpWT; { p,0.05 vs BpCyaA2.doi:10.1371/journal.pone.0008734.g002

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Pam2CSK4, the positive control. TLR4 signaling was also

induced, but the levels of activation were statistically lower with

respect to the ultra pure E. coli LPS, used as positive control.

The core component of TLR signaling is the activation of an IL-

1-like pathway dependent upon the adapter MyD88, leading to

phosphorylation of mitogen activated protein kinases (MAPK)

such as p38, ERK1/2 and SAPK/JNK, and coupling to the

activation of nuclear factor-kB (NF-kB) [32].

Both B. pertussis strains induced the phosphorylation of p38,

ERK1/2, SAPK/JNK, and IkBa, a process that allows the

activation of the NF-kB complex, in MDDC within 5 minutes

after treatment (Figure 4A). The induction of intracellular MAPK

signaling was further analyzed using inhibitors specific for p38

(SB203580) and ERK1/2 (PD98059) and measuring phenotypic

markers and cytokine release (Figure 4B–C). When MDDC were

treated with either BpWT or BpCyaA2, p38 inhibitor induced a

significant inhibition of all maturation markers, in particular

CD83 (reduced by 90%) (Figure 4B), and almost completely

abrogated IL-12p70, IL-23, IL-1b and IL-10 cytokine production

(Figure 4C). The ERK1/2 inhibitor caused a significant reduction

of CD83 maturation marker (by 45%) in the presence of BpWT

with limited effects on cytokine production. When MDDC were

treated with BpCyaA2, the ERK1/2 inhibitor had no significant

influence on phenotypic maturation but elicited some reduction of

cytokine production, particularly of IL-12p70 (inhibited by 70%).

These results demonstrated that B. pertussis exerted intracellular

effects through both p38 and ERK1/2 activation, with the former

having a prominent influence.

Phosphatidyl inositol 3 kinase (PI3K) is activated in response to

various stimuli, including TLR2 engagement [33], thus, consid-

ering the strong involvement of TLR2 activation by B. pertussis

(Figure 3), we investigated its influence in MDDC activation

induced by B. pertussis by using LY294002, a selective inhibitor of

this kinase. The data obtained showed an anti-inflammatory role

of PI3K. While no major differences were observed in phenotypic

maturation in the presence of either BpWT or BpCyaA2

(Figure 4B), this inhibitor caused strong effects on cytokine

production (Figure 4C), in particular a sharply increased level of

IL-23 production (.100% in the case of BpCyaA2) and IL-1b (by

more than 50% with both strains). Conversely, a clear reduction of

IL-10 levels was obtained under these conditions.

B. pertussis CyaA Blocks the Activation of theMyD88-Independent Pathway

The TRIF-dependent signaling pathway downstream of TLR4

may be triggered independently of MyD88 and leads to the

phosphorylation of interferon regulatory factor (IRF)3 and

thereafter to the production of IFNb and IFN-inducible genes,

including IRF1 and IRF8. In a previous study we showed that

BpWT blocks the expression of the p35 subunit of IL-12p70

through the inhibition of IRF1 and IRF8 expression due to CyaA-

dependent intracellular cAMP accumulation [15]. Here, we found

that BpCyaA2 induced in MDDC a higher level of IRF3

phosphorylation compared to BpWT (Figure 5) and we confirmed

that BpWT strongly inhibited the expression of IRF1 and IRF8.

The addition of CyaA to BpCyaA2-treated MDDC was

responsible for the inhibition of both IRF1 and IRF8 expression

(not shown).

p38 and ERK1/2 MAPK Regulate the Induction of Th17and Th1 Polarization

To evaluate the contribution of activation pathways to the

capacity of MDDC to polarize Th responses, we performed

experiments where MDDC were treated with the two Bordetella

strains in the presence of kinase inhibitors and then used to

polarize purified T cells (Figure 6). When the capacity of Bordetella -

treated MDDC to polarize a Th1 response was analyzed by

measuring IFNc, no specific effects were observed by using the

p38 inhibitor, even though p38 inhibition caused a drastic

reduction of all the cytokines measured in MDDC (Figure 4).

The use of ERK1/2 inhibitor clearly and significantly reduced

IFNc production by co-cultured T cells either when MDDC were

treated with BpWT or BpCyaA2. These data indicate that ERK1/

2 -dependent Th1 polarization is induced by B. pertussis indepen-

dently of IL-12p70, which was not induced by BpWT in MDDC.

No specific effect was observed by the use of PI3K inhibitor.

When the capacity to polarize a Th17 response was analyzed

after p38 inhibition, a clear and marked inhibition of IL-17

production by co-cultured T cells (more than 70%) was observed,

either when MDDC were treated with BpWT or BpCyaA2.

When ERK1/2 was inhibited, a significant increase of IL-17

production by co-cultured T cells (by 80%) was observed when

MDDC were treated with BpCyaA2.

PI3K inhibition did not cause any significant modification of IL-

17 production when MDDC were treated with either BpWT or

BpCyaA2.

The capacity to polarize a Th2 response was then analyzed and

we found that p38, ERK1/2 and PI3K inhibition all caused an

enhancement of IL-5 production induced by BpWT- and

BpCyaA2-treated MDDC, although without any statistical

significance as compared to untreated MDDC, suggesting a strong

donor to donor variability in the production of this cytokine.

These results indicate a pivotal role for the ERK1/2 pathway in

the induction of Th1 polarization and the p38 pathway in the

induction of a Th17 response. All the pathways studied are

implicated in the inhibition of the Th2 phenotype.

Discussion

Several studies indicated that B. pertussis infection drives a Th1

immune response, mediated by IFNc production, both in mice [6]

and in humans [34,35]. We recently demonstrated that B. pertussis

induces IL-23 expression in human MDDC and promotes Th1

Figure 3. TLR4 and TLR2 activation. Triggering of TLR4 and TLR2 intransfected HEK293 cells. HEK293/TLR4/p-Nifty2-SEAP and HEK293/TLR2/p-Nifty2-SEAP cells were either untreated (none) or treated withBpWT or BpCyaA2 for 16 h. Positive control (PC) for TLR4 stimulationwas E. coli LPS (0.1 mg/ml) and positive control for TLR2 stimulation wasPam2CSK4 (0.1 mg/ml). SEAP activity in supernatants of cell cultures wasmeasured. Data are reported as fold increase of SEAP activity overuntreated values. Mean expression 6 SE of ten independentexperiments is indicated. * p,0.05 vs none; u p,0.05 vs PC.doi:10.1371/journal.pone.0008734.g003

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Figure 4. Analysis of MyD88 -dependent pathway induction. A MDDC were either untreated or treated with BpWT or BpCyaA2.Phosphorylation of p38, ERK1/2, SAP/JNK and IkB-a was determined at the indicated time-points by Western blot. A single gel was run and blotted todetect phosphorylated proteins and b tubulin to normalize the results. Data are from one representative out of four independent experimentsperformed with MDDC obtained from different donors. B MDDC were treated as in panel A, either in the absence or presence of p38 inhibitor(SB203580), ERK1/2 inhibitor (PD98059) or PI3K inhibitor (LY294002) for 48 h. Results of seven independent experiments performed with MDDCobtained from different donors are expressed as the percent of change of maturation markers (CD80, CD83, CD38) with respect to the correspondingstimulus in the absence of inhibitors. Mean 6 SE of marker expression in MDDC not treated with inhibitors was for BpWT: CD80 (MFI) = 4866; CD83(%) = 2366; CD38 (MFI) = 1864; for BpCyaA2: CD80 (MFI) = 72611; CD83 (%) = 3667; CD38 (MFI) = 4665. * p,0.05 vs control, calculated from theraw data. C MDDC were treated as in panels A and B. Results of ten (IL-12p70), eight (IL-23 and IL-10) and 5 (IL-1b) independent experimentsperformed with MDDC obtained from different donors, measured by ELISA, are expressed as the percent of change of cytokines with respect to thecorresponding stimulus in the absence of inhibitors. Mean 6 SE of cytokine production (pg/ml) in MDDC not treated with inhibitors was for BpWT: IL-12p70 = 060; IL-23 = 310670; IL-1b = 330685; IL-10 = 20346605; for BpCyaA2: IL-12p70 = 374643; IL-23 = 6726140; IL-1b= 600687; IL-10 = 24776593. * p,0.05 vs control, calculated from the raw data.doi:10.1371/journal.pone.0008734.g004

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polarization in the absence of IL-12p70 [15,23]. Here, we add new

evidence showing that B. pertussis biased the host response toward a

mixed Th1/Th17 phenotype through modulation of MDDC

functions, and identified the CyaA and MAPKs as key players in

this process. Indeed, CyaA limited Th1 and enhanced Th17

responses, and the p38 and ERK1/2 MAPK pathways were

involved in the induction of Th17 and Th1 effectors, respectively.

B. pertussis triggers phenotypic maturation of MDDC [15,23].

Here, we broadened our analysis to CD38. We confirmed that

CD38 expression in MDDC was tightly linked to CD83 and IL-

12p70 production [27]. Indeed, CD38, CD83 and IL-12p70 were

all up-regulated when CyaA was absent (Table 1 and Figure 1).

CD38 exerts pleiotropic activities in immune cells and particularly

in DC [36]. Indeed, besides being functionally involved in CD83

expression and IL-12 p70 induction [27], CD38 also ensures

efficient chemotaxis and transendothelial migration driven by CC

chemokine ligand 21 (CCL21) [26]. Thus, the down regulation

exerted by CyaA on CD38 functions can be considered as a

possible escape mechanism adopted by the bacterium to hamper

the host immune response.

Endogenous CyaA expressed by BpWT, or exogenously added to

BpCyaA2 -treated MDDC, limited the production of the cytokines

analyzed. In particular, IL-23 and IL-1b were strongly reduced and

IL-12p70 completely abrogated. MDDC treated with BpWT

induced a Th1 polarized response, characterized by the production

of high IFNc levels (Figure 2). These results were in agreement with

our previous studies [15,23] and confirmed that B. pertussis induces

in MDDC the expression of Th1 promoting factor(s) other than IL-

12p70. As expected, BpCyaA2 -treated MDDC were stronger

inducers of Th1 polarization than BpWT -treated MDDC

(Figure 2), due presumably to the action of IL-12p70.

The relevance of IL-17 activities in response to Bordetella infection

has been pointed out in mice [37–39], and in humans [40,41]. The

present study demonstrated that Th17 polarization was induced ex

vivo by B. pertussis -treated MDDC. Interestingly, IL-17 induced in T

cells by BpWT -treated MDDC was greater than that from

BpCyaA2 -treated MDDC, indicating that CyaA promoted IL-17

production by T cells. However, the capacity to induce a stronger

Th17 polarization appeared to be unrelated to the levels of Th17

promoting cytokines, such as IL-23, IL-1b, and IL-6, that were

expressed at higher levels by BpCyaA2 -treated MDDC. Instead,

the regulatory activity of IL-12p70, induced in MDDC in the

absence of active CyaA, may play a prominent role in inhibiting

Th17 polarization. This would be in agreement with studies

showing that IL-12p70 is detrimental for the expansion of Th17

cells [42]. Overall, the ex vivo polarization studies showed that

CyaA was ultimately responsible for enhancing Th17 and reducing

Th1 immunity induced by B. pertussis.

Figure 5. Analysis of MyD88-independent pathway induction.MDDC were either untreated (none) or treated with BpWT or BpCyaA2.Phosphorylation of IRF3 was determined at the indicated time-points byWestern blot. Data are from one representative out of threeindependent experiments performed with MDDC obtained fromdifferent donors.doi:10.1371/journal.pone.0008734.g005

Figure 6. Effects of kinase inhibition on Th polarization. A MDDC,either untreated or treated with BpWT or BpCyaA2 in the absence orpresence of p38 inhibitor (SB203580), ERK1/2 inhibitor (PD98059) or PI3Kinhibitor (LY294002) for 48 h, were co-cultured with purified T cells. Onday 12, supernatants were collected and secreted cytokines weremeasured by ELISA. Results of four independent experiments performedwith MDDC and T cells obtained from different donors are expressed asthe percent of change of cytokines (IFNc, IL-17 and IL-5) with respect tothe corresponding stimulus in the absence of inhibitors. Mean 6 SE ofcytokine production (pg/ml) in MDDC not treated with inhibitors was forBpWT: IFNc= 901361828; IL-17 = 900668; IL-5 = 71619; for BpCyaA2:IFNc= 983461225; IL-17 = 423694; IL-5 = 2165. * p,0.05 vs control,calculated from the raw data.doi:10.1371/journal.pone.0008734.g006

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TLR4 signaling mediated by B. pertussis was reported to activate

IL-10 production in mice [43] and to be involved in whole cell

vaccine-induced protection through Th1/Th17 induction [39].

Here we demonstrated a low intensity TLR4 and a strong TLR2

activation by B. pertussis (Figure 3). TLR4 engagement by B.

pertussis, although at low levels, was sufficient to activate the

MyD88-independent pathway, as testified by phosphorylation of

IRF3 (Figure 5), a link between TLR4 activation and the induction

of interferon inducible IRF1 and IRF8 necessary for MyD88-

independent IL-12p70 induction [44,45]. Both IRF3 phosphory-

lation and IRF1/IRF8 mRNA transcription were better promoted

by BpCyaA2 than BpWT indicating an inhibition of MyD88-

independent pathway by CyaA, that might represent a further

mechanisms adopted by B. pertussis to dampen the host immune

responses.

The use of MAPK inhibitors demonstrated a decisive role for

p38 in sustaining phenotypic maturation and cytokine production,

while ERK1/2 activity was involved in IL-12p70 production in

BpCyaA2 -treated MDDC (Figure 4B, C). Th1 induction, in the

form of IFNc production by BpWT -treated MDDC still occurred

after p38 inhibition (Figure 6), thus developing independently of

IL-12p70, which is no longer produced in these conditions

(Figure 4C). These data, together with the observation that BpWT

-treated MDDC promoted a Th1 expansion in the absence of IL-

12p70, indicated the presence of a non-canonical Th1 driving

signal induced by B. pertussis. The fact that inhibition of ERK1/2

pathway in MDDC resulted in significant decrease of IFNcproduction by T cells (Figure 6), suggested that the Th1 driving

signal was dependent on ERK1/2 activity.

Thus, two different pathways appeared involved in the Th1

polarization. ERK1/2 signals might be responsible of IFNcinduction by BpWT -treated MDDC in the absence IL-12p70,

while in BpCyaA2 -treated MDDC p38-dependent IL-12p70

production enhances the Th1 profile.

Concerning Th17 polarization, it was dramatically reduced by

the p38 inhibitor (Figure 6). This result may reflect the strong

reduction of IL-1b and IL-23 production caused by p38 inhibition

in MDDC (Figure 4C).

ERK1/2 inhibition induced a significant increase of IL-17

production promoted by MDDC treated with BpCyaA2

(Figure 6), likely due to a sharp inhibition of IL-12p70 production

accompanied by a slight reduction of IL-23 and IL-1b in

BpCyaA2 -treated MDDC, suggesting a negative regulatory role

of IL-12p70 in the development of Th17 immunity [42].

Inhibition of PI3K activity in MDDC increased production of

IL-23 and IL-1b together with a concomitant reduction of IL-10,

suggesting an anti-inflammatory role of this kinase involved in the

regulation of innate responses to microbial pathogens [33].

However, PI3K inhibition did not cause appreciable changes in

Th polarization, apart from an increase in IL-5 levels with

BpCyaA2 -treated MDDC (Figure 6).

The fact that B. pertussis is able to potentiate the Th17 response,

even in the absence of CyaA, might be linked to the presence of

pertussis toxin. We have recently demonstrated that genetically

detoxified Pertussis toxin (dPT) -treated MDDC drive a mixed

Th1/Th17 polarization [41]. Like B. pertussis, dPT activates the

p38 and ERK1/2 pathways in MDDC, regulators of cytokine

expression, and the PI3K pathway, crucial in limiting IL-23 and

IL-1b and enhancing IL-10 production. A major difference from

the data reported here was in T cell polarization by dPT-treated

MDDC, where p38 inhibition markedly reduced the production of

IFNc by co-cultured T cells, reflecting the induction of the

canonical p38 -dependent and IL-12p70 -mediated Th1 polari-

zation. The differences observed in the activation pathway in

MDDC/T cells in B. pertussis and dPT are not surprising

considering the complexity of the interaction exerted by the

bacterium upon MDDC activation.

Previous observations have shown that B. pertussis and B.

bronchiseptica lacking CyaA caused a diminished inflammation

pathology in mice, due to a reduced ability to recruit leucocytes,

primarily neutrophils, suggesting that infiltrating cells are respon-

sible for lung injury [46,47]. It is now possible to re-interpret these

data in the light of the results of this study. Here we have shown

that CyaA ablation resulted in a reduced capacity to promote

expansion of IL-17 producing effectors, and it is well known that

the over-production of IL-17 fosters neutrophil recruitment to the

site of inflammation [48].

In the light of the results depicted here, it is possible to speculate

that CyaA mediates an escape strategy for the bacterium since it

reduces Th1 immunity and increases the Th17 responses thought

to be responsible, when the response is exacerbated, of enhanced

lung inflammation and injury.

Materials and Methods

Ethic StatementThis study was conducted according to the principles expressed

in the Declaration of Helsinki. All blood donors provided written

informed consent for the collection of samples and subsequent

analysis and the blood samples were processed anonymously. The

study was approved by the Review Board of the University La

Sapienza, Rome, Italy.

ReagentsRecombinant Bordetella pertussis CyaA and CyaA* were ex-

pressed and purified as described elsewhere [24,25]. Polymyxin B

and Brefeldin A were purchased from Sigma Chemicals (St. Louis,

MO). Purified E. coli LPS, synthetic bacterial lipoprotein S-[2,3-

bis(palmitoloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-

[S]-lysyl-[S]-lysyl-[S]-lysine X 3CF3COOH (Pam2CSK4), p44-

p42 ERK1/2 inhibitor PD98059, p38 MAPK inhibitor

SB203580, PI3K inhibitor LY294002 were from Cayla -

InvivoGen Europe (Toulouse, France). Human rGM-CSF and

rIL-4 were from R&D Systems (Minneapolis, MN). rIL-2 was

obtained from Roche (Basel, Switzerland). Fluorochrome-conju-

gated anti-human CD3, CD1a, CD14, CD38, CD80, CD83 and

appropriated mouse isotype-matched mAbs were from BD

Biosciences Europe (Erembodegem, Belgium). Rabbit polyclonal

IgG anti-p-p44/42 mitogen-activated protein kinases (MAPK)

(Thr202/Tyr204, ERK1/2), anti-p-p38 MAPK (Thr180/

Tyr182), anti-p-JNK/Stress Activated Protein Kinase (SAPK)

(Thr183/Tyr185), anti-p-I kappa B alpha (IkBa) (Ser32), anti-p-

IRF3 Ab were from Cell Signaling Technology, Inc. (Danvers,

MA). Mouse anti-b tubulin was from Invitrogen (Paisley, UK).

Bacterial Strains and Growth ConditionsB. pertussis strains: Bp18323 (BpWT) (ATCC reference strain 97-

97) and its isogenic Bp18HS19 mutant (BpCyaA2) [15,18] were

inoculated onto charcoal agar plates supplemented with 10% of

sheep blood (Oxoid, Basingstoke, UK) and grown at 37uC for 72 h

to visualize hemolysis and plated again on charcoal agar for 48 h

at 37uC. Bacteria were then collected and re-suspended in 5 ml of

phosphate buffered saline (PBS). Bacterial concentration was

estimated by measuring the optical density at 600 nm and the

suspension adjusted to a final concentration of 109 CFU/ml. The

bacterial concentration was then checked retrospectively by CFU

evaluation of the final bacteria suspension.

B.pertussis and Th1/Th17 Cells

PLoS ONE | www.plosone.org 7 January 2010 | Volume 5 | Issue 1 | e8734

Purification and Culture of MDDCHuman monocytes were purified from peripheral blood of

healthy blood donors (Courtesy of Dr. Ferrazza, ‘‘Centro

Trasfusionale Policlinico Umberto I’’, University La Sapienza,

Rome, Italy) and cultured in RPMI 1640 (GIBCO, Invitrogen),

supplemented with heat-inactivated 10% LPS-screened FCS,

1 mM sodium pyruvate, 0.1 mM nonessential amino acids,

2 mM L-glutamine, 25 mM HEPES (N-2-hydroxyethylpiprazin-

N’-2-ethansulfonic acid), 100 U/ml penicillin, 100 mg/ml strep-

tomycin, all from Hyclone Laboratories and 0.05 mM 2-ME

(Sigma) (hereafter defined as complete medium) in the presence of

GM-CSF (25 ng/ml) and IL-4 (25 ng/ml). After 6 days, immature

MDDC were washed and analyzed by cytofluorometric analysis

for CD1a, CD14 and CD38 expression. Where indicated, MDDC

were treated with ERK1/2 inhibitor PD98059 (100 mM), p38

inhibitor SB203580 (20 mM) or PI3K inhibitor LY294002 (5 mM )

for 1 h prior to B. pertussis treatment. E. coli LPS (100 ng/ml) was

used as positive stimulus to induce MDDC maturation.

MDDC (106 cell/ml) were re-suspended in complete medium

without penicillin and streptomycin (hereafter defined as Bp

medium), and treated with B. pertussis cells as described elsewhere

[23]. After 2h, cells were extensively washed in the presence of

polymyxin B (5 mg/ml) and incubated at 37uC, 5% CO2 for 48 h

in Bp medium with 100 U/ml penicillin and 100 mg/ml

streptomycin added. In a previous study we tested several

bacterium-to-cell ratios, and the 100:1 was chosen as optimal

ratio on the basis of bacterial ability to induce MDDC maturation

without affecting viability [23].

Where indicated, recombinant CyaA (50 ng/ml) or CyaA*

(50 ng/ml) was added at optimal dosage (determined in prelim-

inary experiments) to MDDC cultures, alone or immediately after

BpCyaA2 treatment.

After 48 h, treated MDDC were harvested for immunopheno-

typic analysis and supernatants for cytokine measurement by

ELISA.

Cell LinesHuman epithelial kidney (HEK)-293 cells stably transfected

with human TLR4, MD2 and CD14 (HEK/TLR4) or human

TLR2 (HEK/TLR2) were purchased from InvivoGen; The

HEK/TLR clones were grown in standard Dulbecco’s modified

Eagle’s medium (DMEM) (GIBCO, Invitrogen) with heat-

inactivated 10% LPS-screened fetal calf serum (FCS)

(LAL,1 ng/ml LPS) supplemented with 1 mM sodium pyruvate,

0.1 mM non-essential amino acids, 2 mM L-glutamine, all from

Hyclone Laboratories (Logan, OH), and normocinTM (100 mg/ml,

InvivoGen), in a 5% saturated CO2 atmosphere at 37uC. HEK/

TLR4 culture medium was supplemented with 0.2 mM Blasticidin

(InvivoGen) and HygroGoldTM (50 mg/ml, InvivoGen). HEK/

TLR2 culture medium was supplemented with 0.6 mM G418

Sulfate (InvivoGen). HEK293/TLR cells were transfected with a

plasmid encoding secreted alkaline phosphatase (SEAP) (pNifty2-

SEAP, InvivoGen) as previously described [31].

TLR Signaling AssayThe induction of TLR signaling in HEK/TLR/p-Nifty2-SEAP

clones was assessed by measuring SEAP activity using QUANTI-

BlueTM colorimetric assay (InvivoGen). Briefly, HEK/TLR/p-

Nifty2-SEAP cells with specific antibiotics added, as specified

before, were seeded into 24-well plates at a density of 26105 cells

in 0.5 ml HEK medium per well for 48 h and then cells were

either untreated (none), treated for 16 h with B. pertussis at 100:1

bacterium-cell ratio (the optimal ratio to induce maximal TLR4 or

TLR2 signaling without affecting cell viability), ultrapure LPS

from E. coli or Pam2CSK4. Supernatants (10 ml) were then

transferred to a 96-well plate and incubated at 37uC with

QUANTI-BlueTM (200 ml). SEAP activity was measured by

reading optical density at 655 nm with a 3550-UV Microplate

Reader (BioRad, Philadelphia, PA). Data are reported as the fold

induction of SEAP activity over untreated controls.

Isolation and Polarization of T LymphocytesT cells were purified from peripheral blood mononuclear cells by

negative sorting with magnetic beads (Pan T-cell Kit, Milteniy Biotec,

Auburn, CA). Purity of cell preparations was assessed by cytofluoro-

metric staining [23]. T cells (0.56106) were cultured in complete

medium in 24-well plates (Costar) in the presence of MDDC

(0.56105). On day 5, IL-2 (50 U/ml) was added to the cultures. On

day 12, supernatants were harvested for cytokine measurement.

Immunophenotypic AnalysisCells were washed and re-suspended in PBS containing 3% FBS

and 0.09% NaN3, then incubated with a panel of fluorochrome-

conjugated mAbs (obtained from BD Biosciences, San Jose, CA)

specific for MDDC: anti-CD14, CD1a, CD80, CD83 and CD38;

or specific for T cells: anti-CD3. Isotype-matched antibodies were

used as negative control. Cells were analyzed with a FACScan (BD

Biosciences). Fluorescence data are reported as percentage of

positive cells when treatment induces the expression of the marker

in cells that were negative; median fluorescence intensity (MFI)

was used when treatment increased the expression of the marker in

cells that were already positive.

Determination of Cytokine Levels by ELISATo measure cytokine production, MDDC were cultured in the

presence of the indicated stimuli in 12 ml tubes (Falcon, Becton

Dickinson, Lincoln Park, NJ) at 37uC, 5% CO2. Supernatants were

collected after 48 h, and IL-10, IL-12p70, IL-23, IL-1b and IL-6

production was assessed by ELISA (Quantikine, R&D Systems,

Minneapolis, MN) and IL-23 by Bender MedSystem (Burlingame,

CA) with a sensitivity of 1.0 pg/ml for IL-1b, 0.7 pg/ml for IL-6,

3.9 pg/ml for IL-10, 5.0 pg/ml for IL-12p70 and 20.0 pg/ml for

IL-23. Optical density obtained was measured with a 3550-UV

Microplate Reader (BioRad Philadelphia, PA) at 450 nm.

Cytokines in the supernatants from polarized T cells were

assayed by ELISA specific for IFNc, IL-5 and IL-17 (Quantikine,

R&D Systems). The lower detection limits were 8.0 pg/ml for

IFNc, 3.0 pg/ml for IL-5, 15.0 pg/ml for IL-17.

mRNA Cytokine Expression by TaqMan Real-TimeReverse Transcriptase-PCR Aanalysis

To measure cytokine mRNA expression, TaqMan Real-time

Reverse Transcriptase-PCR (RT-PCR) analysis was used (Applied

biosystems, Foster City, CA). Total RNA was extracted from

MDDC at different time points and reverse transcription was

carried out as previously described [15]. TaqMan assays were

performed according to manufacturer’s instructions with an ABI

7700 thermocycler (Applied biosystems). PCR was performed,

amplifying the target cDNA (IRF1 and IRF8) transcripts and the

b-actin cDNA as endogenous control. Data obtained were

analyzed with PE Relative Quantification software of Applied

Biosystems. Specific mRNA transcript levels were expressed as fold

increase compared to basal conditions.

Western Blot AnalysisMDDC were starved by culturing overnight in culture medium

supplemented with 1% LPS-screened FCS. Cells were then

B.pertussis and Th1/Th17 Cells

PLoS ONE | www.plosone.org 8 January 2010 | Volume 5 | Issue 1 | e8734

stimulated with B. pertussis at different time points and lysed in

RIPA buffer as previously described [40]. Immunoreactive protein

was detected by incubating blots with anti-phosphorylated proteins

(or anti-unphosphorylated b-tubulin as control) overnight at 4uC.

Blots were washed in Tris-buffered saline 0.1% Tween-20,

incubated with horse-radish-peroxidase (HRP)-conjugated goat

anti-rabbit IgG (Bio-Rad Laboratories, Hercules, CA) (to reveal

phosphorylated proteins) or HRP-conjugated goat anti-mouse IgG

(GE Healthcare) (to reveal control proteins) developed with the

enhanced chemiluminescence (ECL) reagents from Pierce

(Rockford, IL).

Statistical AnalysisStatistical descriptive analyses were carried out using the SPSS

Inc (Chicago, IL) statistical package. Differences between mean

values were assessed by two-tailed Student’s t test and were

statistically significant for P values ,0.05.

Acknowledgments

Thanks are given to Nicole Guiso for providing Bp18HS19 strain and to

Roger Parton for critically reading the manuscript. The help and technical

assistance of Angelo Gallina, Davide Flego and Manuela Bianco are

gratefully acknowledged.

Author Contributions

Conceived and designed the experiments: GF FS CMA. Performed the

experiments: GF FS RP MN. Analyzed the data: GF FS CMA.

Contributed reagents/materials/analysis tools: GYCC JGC. Wrote the

paper: GF FS JGC CMA.

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